Materials and methods Cell culture
Materials and methods Cell culture and receptor gene transfection. PC12 bradykinin receptor were purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cDNA for mouse CysLT1 or CysLT2 receptor (mCysLT1 and mCysLT2, subcloned into pcDNA3.0) was kindly gifted by Professor C.D. Funk (University of Pennsylvania, USA). The pcDNA3.0 null vector was purchased from Invitrogen (Carlsbad, California, USA). The receptor cDNA expressing vectors and the null vector were lineared by PvuI and transfected into PC12 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. The permanently transfected PC12 cells were selected with 500μg/ml G418 in DMEM supplemented with 10% FBS, 100U/ml penicillin and 100μg/ml streptomycin. Single-cell subclones were isolated and plated at low density in 24-well plates, so that only a few clones grew per plate and one clone grew per well. Cells were grown for over 2 months in the selection media. The transfected PC12 cells were defined PC12/WT (transfected with null pcDNA3.0), PC12/mCysLT1 (pcDNA3.0/mCysLT1), and PC12/mCysLT2 (pcDNA3.0/mCysLT2) cells. Before experiment, the cells were photographed with a digital camera (Nikon Coolpix 4500, Japan) under a microscope (Nikon Eclipse TS100, Japan), and their sizes were measured with ImageTool software (University of Texas Health Science Center, San Antonio, USA). Reverse transcription-polymerase chain reaction (RT-PCR). To determine the mRNA expressions of CysLT1 and CysLT2 receptors, total RNA was extracted using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. For cDNA synthesis, 2μg total RNA was mixed with 1mM dNTP, 0.2μg random primer, 20U RNasin, and 200U M-MuLV reverse transcriptase in 20μl reverse reaction buffer. The mixture was incubated at 42°C for 60min and then heated at 72°C for 10min to deactivate the reverse transcriptase. PCRs were performed on an Eppendorfed Master Cycler. The reaction conditions were set as follows: 1μl cDNA mixture was reacted in 20μl reaction buffer containing 1.5mM MgCl2, 0.2mM dNTPs, 20pM primers and 1U Taq DNA polymerase. The mixtures were initially heated at 94°C for 2min, followed by 35 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 30s; finally, the reaction was stopped at 72°C for 5min. The PCR products (10μl) were separated by 2% agarose gel electrophoresis. The primer sequences were designed by using Primer Premier software and the specificity of the oligonucleotide primers was verified using the program BLASTN, as following: mouse CysLT1 receptor forward 5′-(+) CAA CGA ACT ATC CAC CTT CAC C-3′ and reverse 5′-(+) AGC CTT CTC CTA AAG TTT CCA C-3′; mouse CysLT2 receptor forward 5′-(+) GTC CAC GTG CTG CTC ATA GG-3′ and reverse 5′-(+) ATT GGC TGC AGC CAT GGT C-3′; rat CysLT1 receptor forward 5′-(+) TCT CCG TTG TGG GTT TCT-3′ and reverse 5′-(+) TAT AAG GCA TAG GTG GTG-3′; rat CysLT2 receptor forward 5′-(+) AGC GTT AGG AGT GCC TGG AT-3′ and reverse 5′-(+) CAA GTG GAT GGT CCG AAG TG-3′. Oxygen glucose deprivation (OGD) or LTDtreatment. PC12 cells were cultured on poly-l-lysine-coated glass cover slides or flasks, and OGD was carried out 24h after culture. For OGD treatment, the culture media were changed to a deoxygenated glucose-free or an oxygenated glucose-containing (for controls) Earle’s balanced salt solution at pH 7.4. Then the cells were transferred to a sealed hypoxic box containing a mixture of 95% N2 and 5% CO2 at 37°C, or normal culture conditions (controls) for 1, 3, and 6h. Montelukast and Bay u9773 were added to the media from 30min before OGD treatment to the end of OGD to determine the effects of antagonists. LTD4 (10−7M) was added into the media for 6 or 48h to determine the effect of an agonist. Cell death analysis. After OGD or LTD4 treatment, the cells cultured on glass cover slides were washed with PBS, stained with Hoechst 33258 (0.01mg/ml) and PI (0.01mg/ml, Sigma–Aldrich, USA) for 10min at 37°C, and then fixed in 4% paraformaldehyde. For each cover slide, 1000–1500 cells were observed under a fluorescence microscope (Olympus BX51, Japan) by an investigator without the knowledge of treatments. After Hoechst 33258 fluorescent staining, normal cells showed a homogeneous staining of their nuclei, and apoptotic cells a deep and asymmetric staining of their nuclei as a result of chromatin condensation and nuclear fragmentation. Necrotic cells were stained with PI (red color). The percentage of necrotic or apoptotic cells was calculated.